1. Technical Field
This application relates to the identification, isolation and characterization of endogenous digitalis-like compounds and their administration as therapeutic agents.
2. Background Art
The Na.sup.+,K.sup.+ -ATPase (E.C.3.6.1.3) is an integral plasma membrane protein responsible for the maintenance of Na.sup.+ and K.sup.+ concentration gradients in all eukaryotic cells. Since Na.sup.+,K.sup.+ -ATPase has a high-affinity receptor for digitalis steroids, it has been postulated that there are endogenous ligands for these receptors which regulate the Na.sup.+,K.sup.+ -pump activity. Indeed, based on their ability to inhibit .sup.3 H!-ouabain binding and Na.sup.+,K.sup.+ -ATPase activity, digitalis-like compounds (DLC) have been shown to be present in the brain Haupert, G. T., Jr. & Sancho, J. M. (1979) Proc. Natl. Acad. Sci. USA 76: 4658-4660; Lichtstein, D. & Samuelov, S. (1980) Biochem. Biophys. Res. Com. 96, 1518-1523; Akagawa, K., et al., (1984) J. Neurochem. 42: 775-780!, heart De Pover, A., et al. (1982) Biochem. Pharmacol. 31: 267-271!, adrenal Tamura, M., et al., (1988) Biochemistry 27: 4244-4253!, plasma Kramer, H. J., et al. (1977) Kidney International 12: 214-219; Gruber, K. A. et al. (1980) Nature 237: 743-745; Moreth, K., et al. (1986) Klinische Wochenschrift 64: 239-244; Dasgupta, A., et al. (1987) Biochem. Biophys. Res. Commun. 148: 623-628!, CSF Halperin, J., et al. Proc. Natl. Acad. Sci. USA 80: 6101-6104; Lichtstein, D., et al. (1985) Brain Res. 325: 13-19! and urine Cloix, J. F. et al. (1987) Can. J. Physiol. Pharmacol. 65: 1522-1527; Goto, A., et al. (1990) Biochem. Biophys. Res. Commun. 173: 1093-1101! of mammals and in the skin Flier, J., et al. (1980) Science 208: 503-505! and plasma Lichtstein, D., et al. (1986) Life Sciences 38: 1261-1270! of toads.
Several substances have been proposed as the DLC, including unsaturated fatty acids Bidard, J. N., et al. (1984) Biochim. Biphys. Acta 769: 245-252; Tamura, M., et al. (1985) J. Biol. Chem. 260: 9672-9677; Kelly, R. A., et al. (1986) J. Biol. Chem. 261: 11704-11711!, hydroxy unsaturated fatty acids Lichtstein, D., et al. (1991) J. Endocrinol. 128: 71-78,!, lysophosphatidylcholines Tamura, M., et al. (1987) Biochemistry 26: 2797-2806!, dopamine Clarkson, E. M. & De Wardner, H. E. (1985) Clinical and Experimental Hypertension Part A, A7, 673-683!, dehydroepiandrosterone sulfate Vasdev, S., et al. (1985) Res. Commun. Chem. Path. and Pharmacol. 49: 387-399!, lignan Fagoo, M., et al. (1986) Biochem. Biophys. Res. Commun. 134: 1064-1070! and ascorbic acid Ng, Y. C., et al. (1985) Biochem. Pharmacol 34: 2525-2530!. However, none of these compounds appears to be the natural ligand of the digitalis receptor of the Na.sup.+,K.sup.+ -ATPase because of their limited specificity and affinity.
Bufodienolides, which resemble the structure of the plant cardiac glycosides, have been identified in the plasma, brain and other tissues of toads Flier, J., et al. (1980) ibid.; Lichtstein, D., et al. (1986) ibid.; Meyer, K. & Kinde, H. (1971) Collection of Toad Venoms and Chemistry of the Toad Venom Steroids. Vol. 2 p. 521-552, in Venomous Animals and their Venoms, Eds W. Bucherl & E. Buckley. New York Academic Press; Lichtstein D., et al. (1991) Biochem. Biophys. Acta. 1073: 65-68!. However, the presence of bufodienolides in mammals has not yet been demonstrated. Recently, ouabain Hamlin, J. M., et al. (1991) Proc. Natl. Acad. Sci. USA 88: 6257-6263! and digoxin Goto, A., et al. ibid.! have been identified in mammalian tissues but their synthesis by mammals has not been demonstrated. Numerous studies have implicated DLC in the regulation of sodium and water homeostasis and pregnancy as well as pathological states such as hypertension and renal and hepatic failure for review see De Wardener, H. E. & Clarkson, E. M. (1985) Physiological Reviews 65: 658-759; Haber, E. & Haupert, G. T. (1987) Hypertension 9: 315-324; Wechter, W. J. & Benaksas, Prog. Drug Res. 1990, 34: 231-260!.
Availability of endogenous digitalis-like compounds and the possibility of their administration as therapeutic agents may result in advantageous treatment of various pathological conditions in which their involvement is implied. Such compounds may be used as cardiotonic agents, increasing the intensity of heart muscle contractions, as vasoactive agents, elevating blood pressure and as natriuretic/diuretic agents, increasing the excretion of sodium into the urine and thus increasing urine volume. In view of the marked involvement of Na.sup.+,K.sup.+ -ATPase in the central nervous system, digitalis-like compounds may also be used as neuromodulating agents.
Moreover, the levels of endogenous digitalis-like compounds in body fluids or tissues may be used for diagnosing said pathological conditions such as hypertension or kidney or hepatic failure.
In search for endogenous digitalis-like compounds, the present inventors have surprisingly found that such compounds are present in human cataractous lens nuclei.
The eye lens is an avascular tissue composed of two different cell types: fiber cells that are essentially free of intracellular organelles that make up the bulk of the lens, and a single layer of epithelial cells on the anterior surface from which the fiber cells are derived Duncan, G. & Jacob, T. J. The Eye (Davson, H., Ed.) pp. 159-206, Academic Press, Orlando, Fla., USA (1984). The anatomy and biochemistry of the lens as well as its development were extensively studied (for review see Duncan, G. & Jacob, T. J. (1984) ibid.; Cotlier E., The Lens in: Adler's Physiology of the Eye (Moses R. A. ed) 277-303, The C. V. Mosby Company, St. Louis (1981)!. Studies of lens plasma membrane ion pumps have indicated that the lens Na.sup.+,K.sup.+ -ATPase could maintain intracellular Na.sup.+ and K.sup.+ concentrations similar to those in other tissues Sen P. C. & Pffifer D. R. (1982) Biochim. Biophys. Acta 693: 33-34!.
Several studies raised the possibility that a Na.sup.+,K.sup.+ -ATPase inhibitor is involved in cataract formation. In 1960, a strain of mice that develops lens opacity shortly after birth was described Nakano K., et al. (1960) Jap. J. Ophthalmol. 14: 196!. Further investigation of the mechanism of the development of this hereditary cataract suggested that an apparent deficiency of Na.sup.+,K.sup.+ -ATPase activity in the lens may be involved Itawa, S. & Kinoshita, J. H. (1971) Invest. Ophthalmol. 10: 504-512!. The continuation of this research led to the isolation of a "cataracteogenic factor" from mice lenses, that inhibits Na.sup.+,K.sup.+ -ATPase activity Fukui H. N., et al. (1978) Exp. Eye. Res. 26: 1-9; Russell P., et al. (1981) Vision Res. 21: 37-39!. It was further demonstrated that the levels of this endogenous inhibitor are elevated in cataractous lens but it is also present in the normal lens Russell, P., et al. (1981) ibid.! The study of Fukui et al. (1978) ibid.! as well as the recent study by Kami and Sakai Kamei, A. & Sakai, H. (1989) Jap. J. Ophthalmol. 33: 348-357! strengthen the possibility that this Na.sup.+,K.sup.+ -ATPase inhibitor is a peptide. However its amino acid composition and sequence have not been determined.